All animals coexist with microbes, often in intimate and specific associations. The indigenous microbiota of mammals plays important roles in the development and function of the digestive and immune systems. Many of the studies on the role of the normal microbiota has been conducted in rodent models, using gnotobiology to investigate morphological and physiological differences between conventionally and sterilely reared (germ-free) animals. While the gnotobiotic mouse has been a powerful system in which to examine this question, it has several limitations. First, the cost and expertise required to maintain this model have limited its use to a handful of research centers worldwide. Second, the mouse model is not amenable to large-scale screens to explore the host factors involved in bacterial-host interactions. In this R21 grant proposal for innovative and exploratory research in digestive diseases and nutrition, we propose to develop the zebrafish, Danio rerio, as a system to study the role of the microbiota in development of the gastrointestinal tract. The experiments proposed will establish the groundwork for a system in which we can explore the molecular genetics of bacterial-host interactions in gut development, taking advantage of all of the tools offered by this model vertebrate host including forward genetics and genomics. Specifically, we propose to ask: 1). What bacteria are normally associated with laboratory-reared zebrafish during development? 2). Can zebrafish be reared germ-free? 3). Are there phenotypic differences between conventionally reared and germ-free zebrafish? 4). Is colonization of germ-free zebrafish with bacteria sufficient to reverse any of the germ-free phenotypes? By establishing this vertebrate model system, we will be able to experimentally address questions important for understanding and treating human diseases associated with imbalances in and inappropriate responses to the indigenous microbiota, such as opportunistic infections, inflammatory bowel disease, and atopic allergies.